Research project molecular targets for diagnosis and treatment of cancer

Research group Tumour Phenotype, Dept. of Molecular Cell Biology

Cyto- and nucleoskeleton behavior during apoptosis

The structures of the cytoskeleton and the nuclear matrix are not only subject to degradation during apoptosis. These structures do also provide structural support and a scaffold for the assembly and activation of different molecular structures that are pivotal in the initiation and execution of the process of programmed cell death.

The proper function and organization of the cyto- and nucleoskeletal structures in cancer cells can therefore influence the efficiency of anti-cancer therapies that are based on the induction of apoptosis. Indeed, the sensitivity for of apoptosis.

In this research line the mechanisms underlying the specific changes in the cyto- and nucleoskeleton during apoptosis are studied. Research is mainly focused on keratins, one of the components of the cytoskeleton. The keratin network is amongst the primary targets during the execution phase of apoptosis.

We have shown that the keratin network also plays a role in sensitizing cells to roscovitine-induced apoptosis by efficient recruitment of DEDD. In cells expressing high levels of DEDD, this protein is diubiquinated and shows a strong association with the cytokeratin scaffold. Cells showing high levels of DEDD are more sensitive to roscovitine-induced apoptosis in contrast to cells expressing low levels of DEDD, although both type of cells show similar procaspase-3 contents. We hypothesize that the DEDD-mediated recruitment of procaspases-3 and -9 leads to an increase in their local concentration, which renders cells more apoptosis-prone. When triggered, these cells respond in a rapid and efficient way to roscovitine-induced apoptosis in contrast to cells expressing low levels of DEDD.

We also showed that the C-terminal cytokeratin 18 peptide, liberated by early caspase-9 cleavage, plays a role in regulating early events during the execution phase of apoptosis. The C-terminal peptide, which shows basic properties, relocates specifically to the nucleoplasm as shown by confocal laser scanning microscopy using a specific antibody to this cytokeratin 18 region and is able to modulate topoisomerase activity in vitro as measured by relaxation of plasmid DNA. Based on our observations, we suggest that translocated C-terminal cytokeratin 18 peptide assists topoisomerase I in the adequate performance of nucleolar processes during the initial phase of apoptosis and indirectly supports protein biosynthesis via regulation of rDNA transcription and rRNA processing.

Role of p21 Waf1/Cip1 during cellular senescence

This research line is focused on the role of p21Waf1/cip1 during treatment induced senescence. Cellular senescence is a backup program for cell death and is characterized by altered gene expression and morphology. Although unable to proliferate, senescent cells interfere with cell growth by the secretion of a variety of growth factors. Using the parental HCT116 wt and the p53-/- and p21-/- knockout cell lines, we showed that p21Waf1/cip1 plays a pivotal role in the induction and maintenance of drug-induced senescence. In contrast to wild type cells the p21-/- knockout cells failed to undergo senescence after low dose treatment with the topoisomerase inhibitors etoposide or campthotecin, the cdk-inhibitor roscovitine was

unable to induce senescence.

Double labeling techniques were developed to detect chromosomal aberrations in combination with either cytoskeletal markers or the presence of incorporated BrdU

In this way we were able to discriminate between cells in the G2-phase of the cell cycle, tetraploid G1-phase cells, binucleated cells or aggregates of two G1-phase cells and we were able to show if genetically aberrant cells are capable of proliferating. We showed that during treatment with low dose topoisomerase inhibitors the number of tetraploid cells increased significantly in the p21-and p53- knockout cells as compared with the wild type cells. Similar observations were made when p53 knockout cells were exposed to short periods of anoxia. Furthermore we showed that these tetraploid cells are still able to proliferate as evidenced by their ability to incorporate BrdU. No obvious difference between wild type and knockout cells with respect to long term survival were observed after low dose drug treatment.

Preliminary results show that combined treatment of low dose topoisomerase inhibitors and the cdk-inhibitor roscovitine prevents the formation of tetraploid cells and senstitizes cells to low dose topoisomerase treatment as evidenced by a decrease long term survival.
 

Selected publications

Schutte B, Henfling M, Ramaekers FCS.
DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis.
Apoptosis. 2006;11: 1561-72.

Targets for lung cancer vaccine development

Lung cancer is an important health problem worldwide. In the past decade, no significant improvement of the life expectancy of lung cancer patients has been achieved. Therefore, alternative treatment protocols that can be used next to the current therapies are highly needed for this type of malignancy. In the LCVAC project, a consortium of five academic and business partners, situated in the Netherlands, Great Britain and Germany, will therefore develop a therapeutic vaccination strategy for lung cancer.

The partners in the LCVAC consortium have combined their expertise in the following projects:

• Production of a vaccine based on cancer cells isolated from patient lung cancers. The cells will be propagated under pharmaceutical conditions and irradiated to eliminate their multiplying potential before use as an autologous vaccine.
• Production of a vaccine based on established lung cancer cell lines that are well characterized and still contain lung cancer specific characteristics.
• Production of a protein-based vaccine developed using the proteomics technology used to identify components in which the lung cancer cells differ from the normal lung cells. These components are being isolated, produced and their properties investigated in detail.

In the past year we identified and characterized NCAM-180 variant using differential expression methods and PCR. The NCAM-180 variant was showing to be associated specifically with small cell lung cancer and showed an upregulated expression pattern in cell lines derived from these

tumors. As a result the variant may be used as a target for diagnosis and therapy. We could show that the NCAM-180 is capable of inducing a

cytotoxic T lymphocyte (CTL) response in mice. Furthermore antibodies to this variant could be raised that are applicable in ELISA and immunocytochemistry.

Grant

EU (6th framework)
Ramaekers et al. LCVAC : new vaccination therapies for lung cancer
2004-2006
Euro 388.000

Research group
Prof. dr. Frans C.S. Ramaekers, project leader
Dr. Bert Schutte
Dr. Anton Hopman
Dr. Ann v.d. Borght
Dr. Jos Broers
Dr. Fons Verheyen 

Technicians
Mieke Henfling
Monique Ummelen
Miriam Kamps 

Students
Tanja Geelen
Jill Hagelstein
Leon Spijkers

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The department of dermatology has a number of research lines embedded within the GROW research institute. Both clinical and molecular biological research is supported within the department, with a strong focus on the genetics of epithelial growth and (malignant) differentiation. The diversity in our research, sustained by a relatively small and nimble group, offers the opportunity to quickly move into promising new fields. In 2006, we published 36 papers in international peer-reviewed journals and 17 in national journals. This includes a number of high-impact papers.

Molecular genetics of inherited skin disease

Our department is a national referral center for the diagnosis and treatment of inherited skin disease and syndromal skin abnormalities. All molecular genetics research in the lab is patient-driven, an approach that is quite successful. Fourteen international papers covering this field of research were published in 2006, all of which appeared in high-impact specialist journals. Results include the identification of a new gene important for nail development, reported in a major genetic journal (Am J Hum Genet).

Functional analysis of lamin mutations causing lipodystrophy

Mutations in the LMNA/C gene that codes for nuclear lamina proteins cause a number of diseases characterized by an altered fat distribution. This includes progeroid syndromes and familial partial lipodystrophy. In an effort to understand the molecular mechanisms underlying the fat dystrophy in these diseases we have a PhD project in close collaboration with the department of molecular and cellular biology. This line has now generated exciting results and the first major paper concerning progeria has been published this year (Hum Mol Genet).

Gap junction biology

We have a strong track record in the identification of diseases caused by mutations in gap junction genes. We recently showed that a novel mutation in the GJA1 gene can cause oculo-dento-digital dysplasia with skin symptoms, an unexpected finding. Our discovery sheds new light on the function of GJA1 in the skin. Using mutant gap junction proteins fused to fluorescent reporter proteins, we are now examining the life cycle of gap junction proteins in animal cells. We also perform functional analysis of gap junctions in cell membranes in an international collaboration. We expect this research line to generate high-impact results.

Clinical oncology

Our department is a national referral center for the diagnosis and treatment of non-melanoma skin cancer and has played a major role in the development of Mohs' micrographic surgery for the treatment of basal cell carcinoma. Also alternatives for treatment of these cancers have been developed such as laser resurfacing. Our significant contribution in this field is reflected in the completion of two PhD theses (J. Ostertag, P. Quaedvlieg). Current research is shifting towards molecular oncology focusing on the elucidation of hereditary factors that predispose to developing basal cell carcinoma and other hair follicle tumors. A first publication in this field is on the cancer syndrome Birt-Hogg-Dubé, that is financially supported by the "profileringsfonds" to resolve the functional role of the involved protein.

Biological activities of retinoic acid metabolism blocking agents

Retinoic acid metabolism blocking agents (RAMBAs) are promising new compounds for the treatment of several inherited and acquired genetic disorders. In 2004 we completed a first clinical trial showing the efficacy of one such compound in psoriasis. The results of a second clinical trial with a RAMBA are ongoing. In collaboration with Barrier Therapeutics, Inc (Geel, Belgium), we have started experiments to explore other potential applications for RAMBAs and similar compounds in vitro.

Awards

Sadhanna Badeloe was elected "Clinician of the Year" in recognition of her contributions to medical training. The award is granted to clinicians whose teaching is judged to be outstanding by the medical students themselve

Grants

First stream:
azM, 4 years, AGIKO equivalent: molecular analysis of laminopathies.
PI: P.M. Steijlen & M.A.M. van Steensel 

GROW, 1.5 years, 10.000 €: mismatch repair defects in multiple basal cell carcinoma.
PI: M.A.M. van Steensel 

azM, 3 years, profileringsfonds, 150.000 €: Deregulation of HIF1 and mTOR as a cause of Birt-Hogg-Dubé syndrome.
PI: M.A.M. van Steensel, B. Wouters 

Third stream:
Stichting de Drie Lichten, 1 year, 6.000 €: Regulation of gap junction gene expression.
PI: M.A.M. van Steensel 

Fourth stream:
Barrier Therapeutics, 50.000 €/year: contract research in compound development
PI: P.M. Steijlen, M.A.M. van Steensel & M. van Geel
 

Research group
Prof. Dr. P.M. Steijlen, project leader
Prof. Dr. J.A. Frank
Dr. M. van Geel
Dr. A. Sommer
Dr. M.A.M. van Steensel 

PhD students
Drs. S. Badeloe
Drs. N. Shahid
Drs. V.L.R.M. Verstraeten
Drs. E. de Zwart-Storm
Drs. D. Kuijpers
Drs. P. Quaedvlieg
Drs. R. Ceulen 

Technicians
Ing. M. Kamps
Ing. R.S. Bladergroen
Drs. M.H. Lenders

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Recent research suggests that the partial resistance in solid tumours to radiotherapy (RT) is not only due to hypoxia, but also in part to expression and activation of EGFR. Activation of EGFR has been shown to cause accelerated proliferation, reduced apoptosis, enhanced capacity for DNA repair and angiogenesis - thus opposing the toxic effects of RT. Therefore, we began to investigate the molecular mechanisms and clinical importance of EGFR signalling in tumor hypoxia and RT. Furthermore, we are also interested in the role of a mutant form of EGFR, called EGFRvIII, which is frequently expressed in many different tumor types, in the responses of carcinoma cells to hypoxia and RT. Four different interconnective research lines are currently ongoing:

1. EGFR regulation under conditions of hypoxia: We have been able to demonstrate that hypoxia induces the expression of EGFRvIII, which significantly improves the clonogenic survival of tumor cells during hypoxia with a strong proliferative response. Thus, EGFRvIII improves the hypoxia tolerance of tumor cells, which indicates a possible selection of EGFRvIII expressing cells under hypoxic conditions. This might contribute to the radioresistant phenotype of hypoxic tumor zones.

2. EGFR responses to RT: The aim is to characterize the role of EGFR and EGFRvIII in the molecular and cellular response to RT. We have found that although EGFRvIII is unresponsive to EGFR ligands, its kinase activity is stimulated by RT. We are analysing the relevant phenotypic changes of different tumour cells following vIII activation, including changes in proliferation, cell survival, and angiogenesis. Our work indicates substantial differences between the EGFR- and EGFRvIII- radiation induced signalling pathways, thus suggesting unique cellular consequences of EGFRvIII expression.

3. EGFR family as biomarkers for RT response in prostate cancer: We are currently analysing the predictive role of EGFR/ EGFRvIII, the MAPK and PI3K pathway in prostate cancer patients treated with RT. For this, we developed a tissue microarray containing paraffin-embedded biopsies of more than hundred prostate cancer patients. The development of phospho-specific antibodies enables us to detect activated signalling molecules. In addition, we will perform hierarchical clustering to investigate an approach to stratifying patients for targeted kinase inhibitor therapy. The detailed investigation of EGFRvIII expression will, in addition, further help us to better understand and to define its role in RT

4. Imaging of EGFR in vitro and in vivo: Recently, we also began to image the EGFR expression with the help of labelled Cetuximab, a specific antibody for EGFR in vitro and in vivo. We found, that Cetuximab also binds to EGFRvIII with as a consequence subsequent internalization. Possible functional consequences for EGFRvIII are being investigated.

Funding

Dutch Cancer Society (KWF); 443.000 Euro; Principle Investigator: Guido Lammering; Title: The role of a variant form of EGFR in radiation responses of human malignant tumors; Period 2006 - 2010

 

Figure 1: Hypoxia induces the expression of EGFRvIII, but not EGFR. Within 24 hours of anoxia (0% oxygen), the expression levels of EGFRvIII increase by at least 4 fold.

Figure 2: Expression of EGFRvIII increases hypoxia tolerance. Over a period of 96 hours of anoxia, the survival of cells expressing EGFRvIII increases by at least 5 fold compared to controls.

Figure 3: The clinically used antibody against EGFR, called Cetuximab binds to EGFR and EGFRvIII causing internalization, as shown by confocal microscopy of fluorescent-green-labelled Cetuximab. A431 cells express the wild-type EGFR, while U373vIII expresses only the EGFRvIII protein.

 

Research group
Dr. Guido Lammering, project leader 

Co-Principal Investigators
Prof.dr. Bradly G. Wouters
Prof.dr. Philippe Lambin 

Co-investigators

Post-doctoral fellows
Dr. Roland Chiu
Dr. Jan Theys
Dr. Marianne Koritzinsky 

PhD students
Hugo Aerts
Younan Li
Ludwig Dubois 

Technicians
Natasja Lieuwes
Barry Jutten 

Students
Ruud Straathoef 

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Genome instability is associated with increased cancer risk, and thus considerable effort has been put into unraveling the mechanisms underlying genome surveillance. Guarding the integrity of DNA are a number of DNA-repair and cell cycle-control systems. Insight into how these pathways become activated is crucially important to the understanding of carcinogenesis and in the development of cancer treatments.

As part of a 4 year KWF funded project that concluded in 2006, we made several significant finding in the mechanism of a distinct pathway that promotes the tolerance of DNA damage during its replication phase. Previous knowledge from lower organisms suggested the requirement for enzymes capable of constructing a chain of ubiquitin molecules linked in a specific manner. We used a novel approach to disrupt the formation of these ubiquitin chains in human cells and found that this caused a significant increase in mutations after exposure to UV light. Several lines of evidence implicate a family of error-prone enzymes, called translesion synthesis polymerases, in the formation of these mutations. Furthermore, we provide the first evidence suggesting that proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, is the relevant target of these chains in human cells. This study was published in the prestigious Public Library of Science and received attention in newspaper articles in the Netherlands and Canada. (Chiu et al., PLoS Genetics. 2(7):e116)

We extended this study to investigate the mutagenic effects of the class of environmental carcinogen called polycyclic aromatic hydrocarbons. We hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLh), causing increased BPDE-induced mutagenicity. Together with Prof. Frederik-Jan van Schooten of NUTRIM, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by contributing to genomic stability. (Langie et al., DNA Repair (accepted))

In a manner analogous to phosphorylation, protein modification with polyubiquitin chains is emerging as an important modifier of many signalling pathways including DNA repair. Our understanding of this process in cellular signalling is limited because we lack knowledge of involved substrates. In collaboration with Prof. Edwin de Pauw at the University of Liege, we developed a novel method to isolate linkage specific ubiquitin substrates. Mass spectrometry based determination of K63-linked ubiquitination substrates revealed several known targets including L27a, EF1A1 and NMD3. In addition we identified 14-3-3Z/S as a new target of these novel chains. This work has been submitted for publication in 2006.

Selected publication

Chiu, R.K., Brun, J., Ramaekers, C., Theys, J., Weng, L., Lambin, P., Gray, D.A, and Wouters, B.G.
Lysine63 poly-ubiquitination protects cells against mutations induced by DNA lesion bypass polymerases.
PLoS Genetics (2006). 2(7):e116.

Figure: We have developed a system to selectively block specific types of ubiquitin chains in human cells. Disruption of ubiquitin chains like via lysine 63 leads to a dramatic increase in mutation frequency which corresponds to an increased recruitment of error prone DNA polymerases (demonstrated by foci formation). These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by contributing to genomic stability.

Research group
Prof. Bradly G. Wouters, projectleader
Dr. Roland Chiu
Prof. Philippe Lambin
Dr. Jan Theys 

Post-doctoral fellows
Dr. Christelle Vreuls 

PhD students
Chantal Ramaekers 

Technicians
Kim Paesmans 

Students
Elena Bardina

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The research of this group is composed of two main themes:

1)
Epigenetic-, Genetic and Expression Profiling of colorectal, ovarian, renal cell and breast cancer, to obtain novel insights in cancer biology and discover diagnostic and prognostic markers.
Re-expression micro-array techniques, using cell lines from colorectal, ovarian an renal cell cancer are used to find candidate tumour suppressor genes, which have been epigenetically silenced by DNA promoter hypermethylation. This has resulted in a sensitive and specific panel of biomarkers for colorectal cancer (CRC), which will be tested in blood and faeces from individuals participating in several national (co-operation with a.o. the VUMC in Amsterdam) and international prospective screening projects. One of these is a local CRC screening project organised by the Dept. of Gastroenterology of the University Hospital Maastricht, performed among employees of several companies in the area of Southern Limburg, and was started in the second half of 2006.

Epigenetically silenced genes are studied for their role in differentiating between serous borderline tumours and serous carcinomas of the ovary, whereas the profile of epigenetically silenced genes in renal cell cancer is being correlated with genetic changes in this tumour type (notably VHL mutations) and with downstream effects along the HIF oncogenic pathway.

A potential role of DNA promoter hypermethylation in the development of breast cancer is studied in normal mammary epithelium, in situ carcinoma and invasive cancers.

The work within this theme will most likely be extended to skin and lung cancer. 

2)
Molecular epidemiologic studies on the role of dietary factors in colorectal cancer carcinogenesis.
In a succesful co-operation with the department of Epidemiology (Prof.dr. Piet van den Brandt, Dr. Matty Weijenberg), the relation between (epi-)genetic alterations and dietary factors in a series of approximately 800 CRC patients derived from the Netherlands Cohort Study (NLCS) is studied. Currently, the following questions are addressed:
-Whether promoter methylation of the caretaker genes O⁶-MGMT, MBD4 and hMLH1 leads to specific alterations in the gatekeeper genes K-ras, APC and ?-catenin and MSI.
-Whether dietary intake of components which provide (folate, vitamins B6 and B12) or degrade (alcohol) methyl donors is associated with promoter methylation of O⁶-MGMT and hMLH1.
-Whether the effect of methyl donor intake on promoter methylation is modified by single nucleotide poly-morphisms (SNPs) in genes involved in methyl donor metabolism, i.e. 5-10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR).

This work will provide new insights in the relationship between promoter methylation of caretaker genes and the occurrence of specific genetic alterations in gatekeeper genes involved in CRC carcinogenesis. In addition, the role of dietary intake of methyl donor providers and -degraders and genetic susceptibility, represented by SNPs in genes involved in methyl donor metabolism, on promoter methylation of caretaker genes is clarified.

This might lead to rational dietary interventions, based on the genetic composition of an individual. 

Figure: SNP analysis for methyl donor metabolising enzymes in colorectal cancer

 

Ongoing funding (type of financing)

Transnational University Limburg (1^st )
ZON Mw (2^nd)
SenterNovem (2^nd)
Dutch Cancer Society (3^rd)
Oncomethylome Sciences (4^th)
 

Selected publications

Derks S, Postma C, Moerkerk PT, Bosch van den SM, Carvalho B, Hermsen MA, Giaretti W, Herman JG, Weijenberg MP, Bruïne de AP, Meijer GA, Engeland van M.
Promoter methylation precedes chromosomal alterations in colorectal cancer development.
Cell Oncol 2006; 28(5-6): 247-257

Sieben NL, Roemen GH, Oosting J, Fleuren GJ, Engeland van M, Prat J.
Clonal analysis favors a monoclonal origin for serous borderline tumours with peritoneal implants.
J Pathol 2006; 210(4): 405-411 

Vogel de S, Engeland van M, Luchtenborg M, Bruïne de AP, Roemen GM, Lentjes MH, Goldbohm RA, Brandt van den PA, Goeij de AF, Weijenberg MP.
Dietary folate and APC mutations in sporadic colorectal cancer.
J Nutr 2006; 136(12): 3015-3021.

Hellebrekers DM, Castermans K, Vire E, Dings RP, Hoebers NT, Mayo KH, Oude Egbrink MG, Molema G, Fuks F, van Engeland M, Griffioen AW.
Epigenetic regulation of tumor endothelial cell anergy: silencing of intercellular adhesion molecule-1 by histone modifications.
Cancer Res 2006; 66: 10770-10777

Research group
Dr. Manon van Engeland, molecular biologist, project leader
Prof.dr. Adriaan P. de Bruïne, pathologist
Dr. Nathalie Sieben, pathologist
Dr. Veronique Winnepenninckx, pathologist 

Post doctoral fellows
Dr. Debby Hellebrekers, molecular biologist 

PhD students
Ingrid Bijsmans
Arjen Cleven
Sarah Derks
Marjolein Lentjes
Veerle Melotte
Marcella Stevens-Baldewijns
Stefan de Vogel 

Technicians
Sandra van den Bosch
Kathleen Daenen
Iris Hendriks-Partouns
Peter Moerkerk
Fiona Schot
Angela Spiertz
Kim Wouters 

Students
Iris van Vlodrop
Dora Menting

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The research of this group in the Department of Epidemiology is mainly based on the Netherlands Cohort Study on Diet and Cancer (NLCS). The NLCS started in 1986 and includes 120,852 women aged 55-69 years. At baseline, cohort members completed a mailed, self-administered questionnaire on dietary habits, and other risk factors for cancer (1). The NLCS is followed up regularly by record linkage to the Netherlands Cancer Registry, the national pathology registry PALGA, the municipal population registries and the causes of death registry (Statistics Netherlands).

Etiology and specific molecular endpoints

In close collaboration with the Department of Pathology (prof de Bruïne, dr van Engeland, dr de Goeij) tumor samples have been collected from 735 cases with colorectal cancer from the NLCS. Mutations in oncogenes and tumor suppressor genes have been determined in this population. Furthermore, it has been investigated whether promoter regions of tumor-suppressor genes have been inactivated by hypermethylation. In several PhD-projects, it was investigated whether dietary and environmental exposures at baseline are associated with specific mutations (see e.g. ref 2) or promoter methylation.

In 2007 three projects were ongoing in this area:
- The association between intake of methyl donors or degraders, several genetic polymorphisms and hypermethylation in colorectal cancer (funded by KWF Dutch Cancer Society)
- The association between alcohol intake, polymosphisms in the genes ADH1 and ADH3 and the risk of colorectal cancer (funded by ERAB)
- The association between indicators of weight balance, hypermethylation of tumor-suppressor genes and the risk of colorectal cancer (funded by WCRF)

In cooperation the Department of Urology of the Nijmegen University, also tumor samples have been collected of 235 cases with renal cell cancer from the NLCS. The association has been studied between several risk factors and the presence of mutation in the von Hippel-Lindau (VHL) gene in renal cell cancer. The sample collection has been moved to the Department of Pathology in Maastricht in 2007 and new projects are being prepared. 

Survival of cancer

Since 2004 the NLCS has permission to collect information on the vital status from the automated municipal population registries GBA and information on the cause of death from Statistics Netherlands. The follow-up of cases with colorectal and renal cell cancer of whom tumor samples are available has been completed using these registries. In 2007, it was investigated whether mutation in the VHL gene was an independent prognostic factor in the survival or renal cell cancer. Projects investigating prognostic factors in colorectal cancer are being set up.

Nutrition and cancer

Several projects are currently conducted on the association between diet and cancer.
In collaboration with and funded by the Dutch Food Authority (VWA) the association is being investigated between dietary acrylamide and risk of cancer. In 2007, the NLCS was the first epidemiological study that found evidence that dietary acrylamide was also a human carcinogen. A high intake of dietary acrylamide was associated with an increased risk of both endometrial and ovarian cancer (3). The association of dietary acrylamide and other types of cancer will also be investigated and further results are expected in 2008.

The incidence of adenocarcinoma of the oesophagus and cardia cancer of the stomach is increasing rapidly. Whether risk factors for these cancers are different from the other cancer subtypes in these organs has not been investigated intensively. Funded by KWF, a study is being conducted in which the association of risk factors like BMI (4), smoking, alcohol consumption, vegetable and fruit consumption and selenium status (measured in toenails clippings) with these cancers is investigated. In this study, also the aetiology of the precursor of oesophageal adenocarcinoma, Barrett's oesophagus, will be investigated. 

Gene-environment interactions

In collaboration with the Department of Health Risk Analysis and Toxicology (prof. van Schooten and dr. van Breda) it was investigated whether DNA could be isolated from toenail clippings. These toenail clippings were provided by approx. 90,000 of the 120,000 NLCS participants at baseline and have been kept in a cellar for 20 years. On average, 2 ?g DNA could be extracted from 10 mg toenail material. A complete genotype profile of 10 polymorphisms was successfully generated for 95% of the DNA samples (amplicon sizes ranging from 92 to 148 bp). This offers new and unique possibilities for genetic epidemiologic studies in a very large biobank in a large cohort with an extensive baseline dataset and follow-up (ref 5).

International collaboration

The NLCS collaborates with a.o. the Pooling Project of prospective studies of Diet and Cancer (coordinated by the Harvard School of Public Health) and the Collaborative Group on Epidemiological Studies of Breast and Ovarian Cancer (coordinated by Oxford University). Many high-impact publications are the result from this collaboration.

Selected publications

Van den Brandt PA, Goldbohm RA, van 't Veer P, Volovics A, Hermus RJJ, Sturmans F.
A large-scale prospective cohort study on diet and cancer in The Netherlands.
J Clin Epidemiol 1990; 43: 285-95.

Weijenberg MP, Lüchtenborg M, de Goeij AFPM, Brink M, van Muijen GNP, de Bruïne AP, Goldbohm RA, van den Brandt PA.
Dietary fat and risk of colon and rectal cancer with aberrant MLH1 expression, APC or KRAS genes.
Cancer Causes Control 2007; 18: 865-879.

Hogervorst JG, Schouten LJ, Konings EJ, Goldbohm RA, van den Brandt PA.
A prospective study of dietary acrylamide intake and the risk of endometrial, ovarian, and breast cancer.
Cancer Epidem Biomar Prev 2007; 16: 2304-2313.

Merry AHH, Schouten LJ, Goldbohm RA, van den Brandt PA.
Body mass index, height and risk of adenocarcinomas of the esophagus and gastric cardia: a prospective cohort study.
Gut 2007; 56: 1503-11.

Van Breda SG, Hogervorst JG, Schouten LJ, Knaapen AM, van Delft JH, Goldbohm RA, van Schooten FJ, van den Brandt PA.
Toenails: an easily accessible and long-term stable source of DNA for genetic analyses in large-scale epidemiological studies.
Clin Chem 2007; 6: 1168-1170.

Scientific staff
Piet van den Brandt
Leo Schouten
Bas Verhage
Adri Voogd
Matty Weijenberg 

Postdocs
Lina Leurs
Kim Smits 

PhD students
Brenda Bongaerts
Janneke Hogervorst
Laura Hughes
Jessie Steevens
Stefan de Vogel 

Technicians
Sacha van de Crommert
Jolanda Nelissen
Conny de Zwart

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Molecular alterations and genomic instability in the development and progression of cancer

Our research group focuses on the identification of genomic and phenotypic alterations involved in the pathogenesis of several epithelial and neuro-endocrine derived tumors in order to assess their relevance to predict their biological behavior and to improve the clinical management of cancer patients. In the past decade, collaboration of our research group with several departments in the academic hospital (azM), including Otorhinolaryngology and Head and Neck Surgery, Neurology, Internal Medicine and Pathology, and with other (inter)national institutes has resulted in a number of studies and PhD theses concerning genotypic and phenotypic aberrations identified during the progression of (pre)malignant lesions, such as bladder, brain and unknown primary cancer. In addition, the group is recognized for its expertise in the development and implementation of sensitive in situ hybridisation assays for genomic analysis of tumors.

Human papillomavirus and carcinogenesis

Our studies on uterine cervical and head and neck (pre)malignancies have shown that fluorescence in situ hybridization (FISH) can be reliably applied to assess HPV integration (fig 1A) as a highly sensitive marker for the transition of precursor lesion to invasive carcinoma. These data suggest that mostly singular integration events occur, which is in agreement with PCR-based assays. HPV integration results in disruption of the E1/E2 open-reading frames of the HPV genome, leading to increased expression of E6 and E7 mRNA and proteins. This is in agreement with our observation that individual integration signals harbour high levels of mRNAs. As a consequence p53 and pRb are downregulated favouring a growth advantage and neoplastic transformation. Expression of E6 also has been found to deregulate the telomerase pathway resulting in an upregulation of the telomerase activity. In a recently started PhD project, we have focused on the gene coding for the RNA component of telomerase (TERC) at 3q26 and showed that genomic integration of oncogenic HPV and gain of the human telomerase gene TERC (fig 1B) appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer. Furthermore we were able to proof that integration of HPV and gain of TERC can occur in DNA diploid cells, suggesting that these events are early during cervical carcinogenesis. We obtained an additional grant (LIOF/PathoFinder) aimed to support the development of a sensitive multiplex PCR assay for HPV typing and chromosomal instability. With this assay we will be able to determine in a single assay the HPV genotype, viral load, physical status of the virus and genomic instability.

In oropharyngeal cancers we also established HPV integration in 40% of cases. In these tumors 3q gains has also been detected. Studies are carried out to unravel the molecular mechanisms underlying HPV driven tumorigenesis and to assess new prognostic markers.

Carcinogenesis of neuro-endocrine tumors

The molecular pathogenesis of neuro-endocrine tumors, including endocrine pancreatic tumors (EPTs), bronchial carcinoids, pheochromocytomas and adrenocortical tumors, is not well understood, and the clinical behavior of these tumors is difficult to predict using current morphological criteria.

Moreover, a substantial proportion of tumors is unresectable and responds unpredictable to palliative therapy. Thus, a better understanding of the molecular

processes underlying the tumorigenesis of neuro-endocrine tumors is required to improve diagnosis, prognosis and therapy. Continuation of a PhD study on human sporadic insulinomas revealed, that a high number of confined chromosomal alterations per tumor (defined as chromosomal instability) as well as loss of telomeric ends were strongly associated with metastatic disease. P53 and cytokeratin 19 (CK19) status proved to be of little value for this purpose, because only rare, individual cases showed a TP53 mutation (together with p53 overexpression) or detectable amounts of CK19. In addition, we showed by arrayCGH that gain of chromosome 9q and loss of 22q13.1-q13.31 are early genetic events in sporadic insulinomas, and that loss of 11q and 22q13.31-q13.32 occur later in the tumor development and are associated with loss of telomeric ends. On basis of these data we have proposed a genetic progression model for these tumors (see figure). Two project applications have been submitted to identify candidate genes on the aberrant chromosomal loci and to develop a multi-target test for discrimination of benign and malignant tumors on basis of chromosomal instability. In (inter)national collaborations, furthermore, arrayCGH has been used to identify chromosomal breakpoints on 1p, the most frequently involved genomic alteration in pheochromocytomas, and beta-interferon has been found a highly potent inhibitor of cell growth in neuro-endocrine tumors, including pancreatic carcinoids and adrenocortical carcinomas.

Selected publications

Jonkers YMH, Claessen SMH, Feuth T, Geurts van Kessel A, Ramaekers FCS, Veltman JA, Speel EJM.
Novel candidate tumor suppressor gene loci on chromosomes 11q23-24 and 22q13 involved in human insulinoma tumorigenesis.
J Pathol 210, 450-458, 2006 (IF: 6.213).

Hopman AHN, Theelen W, Hommelberg P, Kamps MA, Herrington CS, Morrison LE, Speel EJM, Smedts F, Ramaekers FCS.
Genomic integration of oncogenic HPV and gain of the Human Telomerase Gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.
J Pathol 210, 412-419, 2006 (IF: 6.213).
 

Awards

• Speel EJM: Best oral communication, 20^th European Congress of Pathology (received in Paris, France, September 6, 2005; Euro 1,500).
• Y. Jonkers: Best abstract, 3^rd European NeuroEndocrine Tumor Society Congress (received in Prague, Chech Republic, March 24, 2006; Euro 1,500).
• Y. Jonkers: Rene Vogels Stipendium (received in Amsterdam, January 17, 2006; Euro 1,700).
  

Major Funding

Association for Internation Cancer Research:
Identification of genes involved in endocrine pancreatic tumor development and progression (Euro 185,000; PhD position)

Molecular Life Sciences tUL:
Impact of virus integration and chromosomal instability on the development and progression of Human Papillomavirus-related tumors (Euro 200,000; PhD position)
 

Research group
Dr. Anton H.N. Hopman, project leader
Dr. Ernst-Jan M. Speel, propject leader
Prof. Dr. Frans C.S. Ramaekers
Prof. Dr. Hans J. Manni
Prof. Dr. Bernd. Kremer
Prof. Dr. Philippe O. van Trappen
Prof. Dr. Adriaan de Bru?ne
Dr. Fredrik J. Bot
Dr. Els V. Meulemans
Dr. Frank Smedts
Dr. Manon van Engeland
Dr. Robert-Jan van Suijlen
Dr. Arnold Kruse 

PhD students
Drs. Harriet C. Hafkamp
Drs. Yvonne M.H. Jonkers
Drs. V. Ewa Bergshoeff
Drs. Wendy Theelen
Boris Klingenberg
Drs. Jos Straatmans

Technicians
Annick Haesevoets
Sandra M.H. Claessen 

Students
Yvo Kusters
Jeroen Mooren
Sophie Litjens
Jenneke Smeenk
Elly Jonker 

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